Estrogen receptors mediate the diverse effects of estrogen on the cardiovascular system. The two known estrogen receptors, ERalpha and ERbeta, are ligand-activated transcription factors that alter gene expression following hormone binding. Recently, it has become clear that ERs also can regulate gene expression in the absence of estrogen by ligand-independent pathways. Ligand-independent activation of ERalpha results from direct phosphorylation of the ER by MAP kinase. Given the importance of phosphatases in modulating kinase-regulated pathways, we hypothesized that phosphatases also regulate ERalpha transcriptional activity. We now present preliminary data demonstrating that ERalpha binds directly to protein phosphatase 2A (PP2A), a phosphatase known to regulate many central cellular processes, and that ERalpha is a substrate for PP2A. We also show that PP2A and MAP kinase associate with ERalpha in a reciprocal fashion, and exert direct counter regulatory effects on ERalpha phosphorylation and transcriptional activation. These data thus demonstrate a previously unrecognized pathway for regulating the transcriptional activation of ERalpha. In the current application, I propose to test the hypothesis that PP2A-mediated regulation of ERalpha has important physiologic effects in vascular cells, by pursuing the following three specific aims. Specific Aim 1: To characterize the regulation of ERalpha binding to PP2A, Specific Aim 2: To identify a physiologic effect of PP2A-mediated regulation of ERalpha, and Specific Aim 3: To identify ERalpha co-activating proteins, that regulate ligand-independent activation of the receptor. Taken together, the results of these studies will provide insight into a previously unrecognized role for estrogen receptors, in regulating vascular cell biology in the absence of estrogen. These findings may have important implications for post-menopausal women, and for men whose circulating levels of estrogen are low. [unreadable] [unreadable]